2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 1 Development of Complex Curricula for Molecular Bionics and Infobionics Programs within a consortial* framework** Consortium leader PETER PAZMANY CATHOLIC UNIVERSITY Consortium members SEMMELWEIS UNIVERSITY, DIALOG CAMPUS PUBLISHER The Project has been realised with the support of the European Union and has been co-financed by the European Social Fund *** **Molekuláris bionika és Infobionika Szakok tananyagának komplex fejlesztése konzorciumi keretben ***A projekt az Európai Unió támogatásával, az Európai Szociális Alap társfinanszírozásával valósul meg. PETER PAZMANY CATHOLIC UNIVERSITY SEMMELWEIS UNIVERSITY dk_fejlec.gif INFOBLOKK ITK_logo_new_375_v1 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 2 ORGANIC AND BIOCHEMISTRY Membrane transport processes www.se.hu (Szerves és biokémia ) (Membrán transzportok) László Csanády http://semmelweis-egyetem.hu/ Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 3 www.se.hu http://semmelweis-egyetem.hu/ Lecture objectives At the end of the presentation the participant will be able to 1. describe the structural background of compartmentalization in living cells 2. understand the phenomenon of osmosis 3. interpret the directionality of transport processes based on thermodynamics 4. differentiate passive from active, primary from secondary, and electrogenic from electroneutral transport 5. calculate equilibrium conditions for various types of transport processes Biochemistry: Membrane transportprocesses 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 4 www.se.hu http://semmelweis-egyetem.hu/ •phospholipid bilayer ¦polar headgroups toward aqueous phase ¦occluded hydrophobic layer ¦fluid mosaicmodel (lateral diffusion) •membrane proteins ¦integral ¦periferal Membrane 1. Structural organization of biological membranes Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 5 www.se.hu http://semmelweis-egyetem.hu/ 2. Membrane permeability •Only lipid-soluble, nonpolar substances can cross the membrane by simple diffusion: O2, N2, NH3, CO2, fats, lipid-soluble drugs •Substances that cannot traverse the lipid bilayer: H2O, ions(K+, Na+, Ca2+, Cl-), water-soluble organic compounds (carbohydrates, aminoacids, nucleotides) These substances can be translocated only with the help of specialized transport proteins! Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 6 www.se.hu http://semmelweis-egyetem.hu/ 3. Compartments •extracellularspace •cytoplasm •endoplasmicreticulum (ER) •mitochondrialmatrix •other cellorganelles 14_2 Biochemistry: Membrane transport processes Compartments 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 7 http://semmelweis-egyetem.hu/ 4. Concentration gradients between individual compartments extracellular(mM) cytoplasmic(mM) Na+14014 K+4140 Ca2+110-4 Cl-1105 cytoplasmic(nM) mitochondrial(nM). H+803 cytoplasmic(mM) ER (mM). Ca2+10-41 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 8 www.se.hu http://semmelweis-egyetem.hu/ 5. Osmosis Solvent moves across a semi-permeablemembrane from the more dilute solution towards the more concentrated solution. Semi-permeable: Allows the small solvent molecules, but not the larger solute molecules to pass. osmosis7 arrow 5.1. The phenomenon of osmosis Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 9 www.se.hu http://semmelweis-egyetem.hu/ osmosis1 5.2. Osmotic pressure The pressure (.) required to stop osmosis. .= cosmRT cosm=osmotic concentration (total molar concentration of all dissolved particles) .is a colligative property: it depends only on the concentration, not the chemical nature, of the solute. Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 10 www.se.hu http://semmelweis-egyetem.hu/ 5.3. Osmotic concentration and cell volume regulation cosm(i.c.)cosm(e.c.) cell expands RBCs Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 11 www.se.hu http://semmelweis-egyetem.hu/ 5.4. Osmotic properties of electrolytes Electrolytes are substances which form ions when dissolved in water. Examples: .salts (NaCl ›Na++ Cl-) .acids (HCl ›H++ Cl-) .bases (NaOH › Na++ OH-) Ions are hydrated in solution –this is energetically favourable (exothermic): the enthalpy of hydration counteracts the input of heat required to break the crystal lattice apart (lattice enthalpy). Dissolving_a_lattice Na_hydration_shell Biochemistry: Membrane transport processes Weak electrolytes:partiallyionize in H2O •weak acids: CH3COOH H++ CH3COO- •weak bases: NH3+ H2O NH4++ OH- TÁMOP –4.1.2-08/2/A/KMR-2009-0006 12 www.se.hu http://semmelweis-egyetem.hu/ Strong electrolytes:completelyionize in H2O .salts: NaCl ›Na++ Cl- .strong acids: HCl ›H++ Cl- .strong bases: NaOH ›Na++ OH- Non-electrolytes:do notionize in H2O •alcohols: ethanol, glycerol •thiols: mercaptoethanol •sugars: glucose, fructose Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 13 www.se.hu http://semmelweis-egyetem.hu/ Ionic dissociation affects colligative properties by increasing the concentration of free solute particles: Osmotic pressure: .=(i.c).RT Exercise: intracellular osmolarity is ~0.3 osmol/l. What is the concentration of an isotonic NaCl, and CaCl2 solution? For NaCl i =2 .(2·c)=0.3 M .c=0.15 M. For CaCl2i =3 .(3·c)=0.3 M .c=0.1 M. 1 mol NaCl ›1 mol Na++ 1 mol Cl- 1 mol CaCl2›1 mol Ca2++ 2 mol Cl- Van't Hoff factor (i) 2 3 .1 1 mol CH3COOH 1 molH++1 mol CH3COO- Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 14 www.se.hu http://semmelweis-egyetem.hu/ Eq_1 6. Thermodynamics of transport processes 6.1. Free energy of substances in solution(J/mol) (electrochemicalpotential, partial Gibbs potential) Go=standard free energy(J/mol) R = 8.31 Jmol-1 K-1 F = 96500 C/mol T = 310 K (37oC) in a mammalian organism z =number of elementary charges of solute(-1, 0, 1, 2, 3, etc.) .=electricalpotential (V) c = concentration of solute inmol/l (check unit of measurement!) Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 15 www.se.hu http://semmelweis-egyetem.hu/ Eq_3 Eq_2 A substance is at equilibrium between two compartments (compartment 1and compartment2), ifG1= G2: 6.2.1. Uncharged substances(z=0) at equilibrium. 6.2. Equilibrium Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 16 www.se.hu http://semmelweis-egyetem.hu/ Eq_5 Eq_4 6.2.2. Charged substances(z.0) at equilibrium (Nernst equation) At body temperature: (where the"reversal potential" Vrev is .1-.2at equilibrium) Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 17 www.se.hu http://semmelweis-egyetem.hu/ Under resting (static) conditions: •substances for which the membrane is permeable must be at thermodynamic equilibriumbetween compartments (otherwise the substance would flow spontaneously into the compartment which provides a lower free energy...) •substances for which the membrane is not permeable can be distributed between compartments in a way that is far from thermodynamic equilibrium Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 18 www.se.hu http://semmelweis-egyetem.hu/ 6.2.3. Examples Plasmamembrane: Under resting conditions permeability forK+ishigh. [K+]e= 4 mM [K+]i= 140 mM .K+is near equilibrium .Vrev= -93 mV .resting membrane potential (Vm .-90 mV) } Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 19 www.se.hu http://semmelweis-egyetem.hu/ Plasmamembrane: Under resting conditions permeability forNa+islow. [Na+]e= 140 mM [Na+]i = 14 mM .Na+is not at equilibrium, it experiences a large thermodynamic driving force .Vrev= +60 mV >>resting membr. potential (Vm .-90 mV) } Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 20 www.se.hu http://semmelweis-egyetem.hu/ A transport process down the electrochemical gradient ("downhill", spontaneous) Why do many substances require specialized transport proteins for passive transport? For kinetic reasons: transport proteins can attain transport rates orders of magnitude larger than that of simple diffusion through the lipid bilayer. 7. Passivetransport Downhill1 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 21 www.se.hu http://semmelweis-egyetem.hu/ 3_cartoons_En Types of passive transporters: •Ion channels •Aquaporins •Uniporters Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 22 www.se.hu http://semmelweis-egyetem.hu/ 7.1. Ion channels Transmembrane protein pores 7.1.1. Gating: the gate opens/closes the pore •a stochastic process, which occurs on the time scale of protein conformational changes (102-104 openings-closings / sec) •diverse cellular signals regulate the probability that the gate is open (e.g., membrane potential –voltage-gated channels, binding of extracellular ligand –ligand-gated channels, phosphorylation, etc.) channel_cartoon Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 23 www.se.hu http://semmelweis-egyetem.hu/ channel_cartoon 7.1.2. Permeation: ion flux through the open pore •when the gate is open, the pore conducts only one (or a few) types of ions(selectivity; e.g., Na+-, K+-, Ca2+-, Cl--, cation-channels) •ions flow passively, down their electrochemical gradient •ion throughput rate can approach 108ion/s •throughput rates saturate at high ionic concentrations Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 24 www.se.hu http://semmelweis-egyetem.hu/ channel_cartoon 7.1.3. Cellular consequences of ion channel function 7.1.3.1. Determination of membrane potential •the activity of ion channels determines the conductivity of the membrane for currents carried by various ions(gNa+, gK+, gCl-) •the magnitude of the currents carried by individual ions is ,whereVXis the reversal potential for ion X •the membrane potential is at rest if the transmembrane currentssum to zero,i.e., if Eq_6 Eq_7 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 25 www.se.hu http://semmelweis-egyetem.hu/ Thus, at rest I.e., the membrane potential is a weighted average of the ionic reversal potentials (Vi), the weights given by the conductivities for the individual ions (gi). Because ion-flux through open channels does not significantly affect the ion concentrations (see 10.1.3.), Viremain relatively constant. I.e., membrane potential changes reflect changes in gi. Eq_8 channel_cartoon Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 26 www.se.hu http://semmelweis-egyetem.hu/ 7.1.3.2. Vectorialion transport (e.g. "transepithelial", across an epithelial layer) •epithelial cells can form tight monolayers in which cells are linked via tight junctions •vectorial ion flow across such epithelia involve active transporters in the basolateral membrane to set up trans- epithelial gradients, and ion channels on the apical surface to allow passive ion flow across the apical membrane Transepithelial_transport_annotated Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 27 www.se.hu http://semmelweis-egyetem.hu/ 7.2. Aquaporins •Transmembrane protein pores selectively permeable to water •The driving force for water flux is osmosis (water flows passively towards the compartment with a higher osmolarity) Osmolarity(osmol/l): Total concentration of all dissolved particles(cosm=.ici) E.g.: osmolarity of a 140 mM NaCl solution is280 mosmol/l ([Na+]=140 mM, [Cl-]=140 mM) aquaporin_cartoon Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 28 www.se.hu http://semmelweis-egyetem.hu/ Osmotic pressure: The hydrostatic pressure required to stop osmosis (.osm= cosmRT) E.g.: the osmotic pressure of a 140 mM NaCl solution at room temperature is700 kPa (= 7 atm) •The absence or presence of aquaporins fundamentally determines the physiological consequences of vectorial transport: ¦vectorial transport in the absence of aquaporins .concentration gradients without volume changes ¦vectorial transport in the presence of aquaporins .volume changes without concentration gradients aquaporin_cartoon Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 29 www.se.hu http://semmelweis-egyetem.hu/ Biophysics of vectorial salt-water transport: a concerted operation of ion channels and aquaporins Transport_a iThe lipid bilayer is impermeable to both ions and water. Transport_b iCl-permeability requires the presence of Cl-channels or transporters Transport_c => the direction of Cl-transport is determined by the electrochemical driving force. Transport_d iWater permeability requires the presence of aquaporin water channels; water follows ions by osmosis across water-permeable membranes. Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 30 www.se.hu http://semmelweis-egyetem.hu/ 7.3. Uniporters •Transmembrane proteins •Catalyze facilitated diffusion: substrate flows down its electrochemical gradient •Substrate throughput rate follows the time scale of protein conformational changes (102-104/s) •Throughput rate saturates at high substrate concentrations uniporter_cartoon_En Facilitated_transport Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 31 www.se.hu http://semmelweis-egyetem.hu/ uniporter_cartoon_En •Specificity: only one type of substrate transported •Examples: ¦Glut-1 (glucose transporter in red blood cell membrane) ¦Glut-5 (fructose transporter in brush border membrane) ¦CAT (cationic amino acid transporter) Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 32 www.se.hu http://semmelweis-egyetem.hu/ A transport process against the electrochemical gradient ("uphill", non-spontaneous) •Primary active transport: "Uphill" transport is directly coupled to ATP hydrolysis •Secondary active transport: "Uphill" transport is coupled to "downhill" transport of another substance (coupled transport) E.g.: symport (cotransport), antiport (exchange) 8. Active transport csorlo Csiga Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 33 www.se.hu http://semmelweis-egyetem.hu/ 8.1. Primary active transport processes Ultimately, these transporters are responsible for the establishement of all transmembrane gradients. •P-type ATPases: Na+-K+-ATPase, Ca2+-ATPase •V-typeATPases: vacuolarH+pumps •F-typeATPases: mitochondrialF1-Fo-ATPase •ABC ATPases: MDR, MRP, etc. Biochemistry: Membrane transport processes Na-K_ATPase 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 34 www.se.hu http://semmelweis-egyetem.hu/ 8.1.1. Na+-K+-ATPase(Na+-K+pump) •Present in the plasma membrane of all animal cells •Pumps out 3Na+ionsin exchange for2K+ions, at the expense of hydrolysis of 1 ATP •Responsible for maintaining theNa+andK+gradient,and thereby the resting membrane potential Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 35 www.se.hu http://semmelweis-egyetem.hu/ 8.1.2. Ca2+-ATPase(Ca2+pump) Important for maintaining low intracellular[Ca2+] •SERCA (ER membrane): Pumps 2 Ca2+ions from the cytosol into the ER in exchange for 2 H+ions, at the expense of hydrolysis of 1 ATP •PMCA (plasma membrane) : Pumps out 1Ca2+ion from the cytosol in exchange for 2 H+ions, at the expense of hydrolysis of 1 ATP SECRA-PMCA Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 36 www.se.hu http://semmelweis-egyetem.hu/ 8.1.3. VacuolarH+pumps •Present in the membranes of synapticand secretory vesicles •They acidify the lumen of the vesicles at the expense of ATP hydrolysis V-ATPase Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 37 www.se.hu http://semmelweis-egyetem.hu/ 8.1.4. F1-Fo-ATPase •Present in the inner mitochondrial membrane •Physiological role: lets 3H+-s flow into the matrix(downhill transport), while it synthesizes 1 ATP from1 ADP + P •Reversible: whenthe thermodynamicgradients are reversed, pumps out 3 H+-s from themitochondrialmatrixat the expense of hydrolysis of 1 ATP F1-F0-ATPase Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 38 www.se.hu http://semmelweis-egyetem.hu/ 8.1.5. ABC ATPases •Transport mostly lipophylic substances at the expense of ATP hydrolysis •Transport mechanism: substrate likely approaches the protein from the membrane by lateral diffusion.flippinginto the opposingmonolayer (flip-flop mechanism) •Examples: bile acid transporters, transporters that cause multidrug resistance ABC-ATPase Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 39 www.se.hu http://semmelweis-egyetem.hu/ NHE-1 8.2. Secondary active transporters (coupled transport) 8.2.1. Na+/H+-exchanger(NHE) •Present in the plasma membrane •Allows 1Na+ion to enter thecytosol (downhill transport), while it extrudes 1 H+ion (uphill transport, see 10.2.2.) •Protects the cell against acidification Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 40 www.se.hu http://semmelweis-egyetem.hu/ 8.2.2. Na+/Ca2+-exchanger(NCX) •Present in the plasma membrane •Allows 3Na+ions to enter the cytosol(downhill transport), while it extrudes 1 Ca2+ion (uphill transport) •Important for maintaining low intracellular[Ca2+] NCX Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 41 www.se.hu http://semmelweis-egyetem.hu/ 8.2.3. Na+-glucosecotransporter (SGLT1) •Located in the apical membrane of intestinal epithelial cells •Mediates absorption of glucose from the gut •Allows 2Na+ions to enter the cytosol(downhill transport), while it also imports1 glucose (against concentration gradient) SGLT-1 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 42 www.se.hu http://semmelweis-egyetem.hu/ 8.2.4. HCO3--Cl-exchanger (AE1, Band 3) •Located in the red blood cell (RBC) membrane and the kidney •Important for blood CO2transport and renal acid secretion •Exchanges 1 Cl-ion for 1 HCO3-ion Band_3 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 43 www.se.hu http://semmelweis-egyetem.hu/ •Mechanism of CO2transport in the blood: In RBCs the AE1 is close to its thermodynamic equilibrium – small, tissue specific environmental changes determine the direction of its operation. In peripheral tissues RBCs take up CO2and release HCO3-, in the lung they take up HCO3-and release CO2–as a result CO2in the blood plasma is replaced by the more soluble HCO3-. Hamburger_shift Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 44 www.se.hu http://semmelweis-egyetem.hu/ •Concentration gradients of transported substrates •Membrane potential: "Electrogenic" transportprocesses are sensitive to membrane potential Electrogenic: transport cycle results in net charge transfer Electroneutral: does not result in net charge transfer •ATP-, ADP-, andP-concentration: Important for primary active transport processes See numerical exercises(10.). 9. Factors that determine the direction of active transport Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 45 www.se.hu http://semmelweis-egyetem.hu/ 10. Numerical exercises 10.1. Calculation of membrane potential 10.1.1. Membrane conductance values of a resting neuron are (in relative units) gK+: gNa+: gCl-= 1: 0.005: 0.1. ([Na+]e=140 mM, [Na+]i=14 mM, [K+]e=4 mM, [K+]i=140 mM, [Cl-]e=110 mM, [Cl-]i=5 mM.) What is the resting membrane potential? The reversal potentials for the three ions are (Nernst equation): VK=-93 mV, VNa=+60 mV, VCl=-81 mV. Hence Eq_9 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 46 www.se.hu http://semmelweis-egyetem.hu/ 10.1.2. At the peak of the action potential, due to opening of Na+channels, relative conductances are altered as follows: gK+: gNa+: gCl-= 1: 20: 0.1. What is the membrane potential at the peak of the action potential? Eq_10 Using the new conductance values as weight factors Biochemistry: Membrane transport processes 10.1.3. Depolarization was caused by Na+influx. How much did intracellular[Na+] increase, if the cell is spherical with a diameter of20 µm, and the specific capacitance of the membrane is0.01 pF/µm2? From the data the cell volume is the cell surface is 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 47 http://semmelweis-egyetem.hu/ Eq_11 Eq_12 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 48 www.se.hu http://semmelweis-egyetem.hu/ hence the total capacitance is The imported charge is How many moles of Na+ions does this charge correspond to? Eq_13 Eq_14 Eq_15 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 49 www.se.hu http://semmelweis-egyetem.hu/ What is the change in concentration due to this amount of ions? Thus,intracellular[Na+] increases from 14 mM to 14.0045 mM. The activity of ion channels can alter the membrane potential without significantly affecting ionic concentrations! Eq_16 Eq_16 Biochemistry: Membrane transport processes 10.2. Na+/H+exchanger: electroneutralsecondaryactivetransport 10.2.1. Under what conditions is the transporter at equilibrium? The catalyzed reaction:1 H+i+ 1 Na+e› 1 H+e+ 1 Na+i If.G<0, the process goes in the forward direction, if.G>0, the process goes in the reverse direction, if.G=0, there is no nettransport. 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 50 www.se.hu http://semmelweis-egyetem.hu/ NHE-1 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 51 www.se.hu http://semmelweis-egyetem.hu/ The free energy change for the above reaction: Eq_17 NHE-1 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 52 www.se.hu http://semmelweis-egyetem.hu/ Eq_19 Eq_18 (electroneutral.does not depend on membrane potential). In the cell[H+]e=10-7.4M, [H+]i=10-7.1M, [Na+]e=140 mM, [Na+]i=14 mM. Thus, In a resting cell the transport cycle goes in the forward direction. BecausezH=zNa=1, Eq_20 Eq_20 Eq_20 Eq_20 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 53 www.se.hu http://semmelweis-egyetem.hu/ 10.2.2. Why is this transporter necessary? From the Nernst equation the reversal potential forH+is Vrev= –18 mV. The resting membrane potential is far more negative(approximately–90 mV),therefore, the thermodynamic driving force forH+is inward directed, and would eventually cause acidification of the cell. Note:On the other hand, theNa+/H+exchangerwould reach equilibrium onlyat [H+]i=10-8.4M (i.e., upon alkalinization of the cytosol!). Therefore, the activity of theNa+/H+exchanger is regulated: it is inactive at pHi.7, and is activated only in response to acidification of the cytosol. Biochemistry: Membrane transport processes 10.3. Na+/Ca2+exchanger: electrogenicsecondaryactivetransport 10.3.1. Under what conditions is the transporter at equilibrium? The catalyzed reaction:1 Ca2+i+ 3 Na+e› 1 Ca2+e+ 3 Na+i If.G<0, the process goes in the forward direction, if.G>0, the process goes in the reverse direction, if.G=0, there is no nettransport. 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 54 www.se.hu http://semmelweis-egyetem.hu/ NCX Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 55 www.se.hu http://semmelweis-egyetem.hu/ Eq_21 The free energy change for the above reaction: NCX Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 56 www.se.hu http://semmelweis-egyetem.hu/ Eq_22 (electrogenic.membrane potential sensitive). SincezCa=2 andzNa=1, Eq_23 Eq_23 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 57 www.se.hu http://semmelweis-egyetem.hu/ In the cell[Ca2+]e=10-3M, [Ca2+]i=10-7M, [Na+]e=140 mM, [Na+]i=14 mM. Thus, the reversal potential of the transporter isVrev=-60 mV. The resting membranepotential is more negative (~–90 mV),i.e.,Vm0, the process goes in the reverse direction, if.G=0, there is no nettransport. Na_K_pump 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 60 http://semmelweis-egyetem.hu/ Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 61 www.se.hu http://semmelweis-egyetem.hu/ The free energy change for the above reaction: Eq_25 Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 62 http://semmelweis-egyetem.hu/ Na_K_pump Eq_27 Eq_27 Eq_26 SincezNa=zK=1, Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 63 www.se.hu http://semmelweis-egyetem.hu/ The standard free energy change for ATP hydrolysis is Na_K_pump Eq_28 Eq_29 Eq_27 Therefore, at body temperature, Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 64 www.se.hu http://semmelweis-egyetem.hu/ Na_K_pump In the cell[Na+]e=140 mM, [Na+]i=14 mM, [K+]e=4 mM, [K+]i=140 mM, [ATP]i=5 mM, [P]i=5 mM, [ADP]i=10-3M (check unit!!). Thus, Vrev=-131 mV. The resting membrane potential is more positive(~–90 mV), i.e., Vm>Vrev, and.G<0. Therefore, under resting conditions the transport cycle proceeds as written:theNa+/K+pump pumpsNa+ions out of the cell. Biochemistry: Membrane transport processes Eq_30 Eq_32 Eq_32 Eq_31 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 65 www.se.hu http://semmelweis-egyetem.hu/ 10.5. Transport processes that involve bicarbonate (HCO3-): Due to the equilibrium CO2(aq) + H2O - H2CO3- HCO3-+ H+, concentrations [H+], [HCO3-], and [CO2(aq)] in solution are always related to each other through the following equilibrium equation: 10.5.1.What is the reversal potential for HCO3-, if pHe=7.4, pHi=7.1, and CO2can diffuse freely through the membrane? Because and we obtain Biochemistry: Membrane transport processes Eq_33 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 66 www.se.hu http://semmelweis-egyetem.hu/ Substituting into the Nernst equation we get Note, that Vrevfor HCO3-is always identical to Vrevfor H+! 10.5.2.The Na+/HCO3--Cl-/H+exchanger transports 1 Na+and 1 HCO3-ion in exchange for 1 Cl-and 1 H+. What is the direction of the transport under physiological conditions? ([Na+]e=140 mM, [Na+]i=14 mM, [Cl-]e=110 mM, [Cl-]i=5 mM, pHe=7.4, pHi=7.1) Let us write up the transport cycle in an arbitrary direction. Biochemistry: Membrane transport processes Eq_35 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 67 www.se.hu http://semmelweis-egyetem.hu/ The catalyzed reaction: 1 Na+e+ 1 HCO3-e+ 1 Cl-i+ 1 H+i› ›1 Na+i+ 1 HCO3-i+ 1 Cl-e+ 1 H+e Because the process is electroneutral, the free energy change for the above reaction is: Biochemistry: Membrane transport processes Eq_38 Eq_37 Eq_36 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 68 www.se.hu http://semmelweis-egyetem.hu/ Because we obtain I.e., Substituting the data into the left-hand side of this equation we obtain 0.55 which is smaller than 1, i.e., .G<0. Thus, under the specified conditions the transport cycle proceeds as written, moving Na+and HCO3-into the cell, while extruding Cl-and H+. Biochemistry: Membrane transport processes 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 69 www.se.hu http://semmelweis-egyetem.hu/ 11. Recommended literature Orvosi Biokémia (Ed. Ádám Veronika): pp 421-447 732895_4 Biochemistry: Membrane transport processes Atkins_1 2011.09.13.. TÁMOP –4.1.2-08/2/A/KMR-2009-0006 70 www.se.hu http://semmelweis-egyetem.hu/ Fizikai kémia I.(Ed. P.W. Atkins): pp 152, 209-233